PhyNexus                                              Technical Note                             
PhyNexus PhyTip 5K Gel Filtration                                                                                         ...
PhyNexus PhyTip 5K Gel Filtration                                                                             Technical No...
PhyNexus PhyTip 5K Gel Filtration                                                                                   Techni...
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PhyNexus Desalting Columns


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PhyNexus columns can be used for desalting or size exclusion by gravity or on an automated liquid handling platform

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PhyNexus Desalting Columns

  1. 1. PhyNexus Technical Note PhyTip® 5K Gel Filtration column for automated high throughput desalting & buffer exchange of functional proteins High Performance: ● Remove >95% of salts with >80% yield of protein ● Retain protein functionality ● <10% CV in volume and concentration ● Ready for downstream assay Automated desalting/buffer exchange of functional proteins: ● Fully automated process with MEA Personal Purification System ● 12 samples desalted/buffer exchanged in ~30 minutes. High throughput format: ● Available in 96 sample format for high throughput processingIntroductionFor biopharmaceutical companies whose focus is preparing large numbers of potential leads thaton antibody and recombinant protein molecules, are ready for functional assays without the needthere is an increasing desire to perform more for scale-up.screens of potential leads where the datagenerated are more physiologically relevant to the In many cases differing elution conditions areissues of potency, toxicity, and other factors. required for various downstream functionalThese assay formats all require that the antibodies assays. Furthermore, the final purificationand recombinant proteins are well purified, product may require an additional preparationenriched and functional. By utilizing high- step to either reduce toxic buffer salts or toperformance micro scale functional protein completely exchange buffer conditions. Thisseparations, it is now possible to obtain more latter step can now be achieved through fullyphysiologically relevant data at the high-content automated desalting and buffer exchange withscreening step and thus make the decision-making PhyTip 5K gel filtration columns used inpower available at the earliest stages in the conjunction with the MEA Personal Purificationdiscovery process. PhyNexus PhyTip column System. These unique columns allow for hightechnology has been developed for high- recovery of the functional antibody of interestthroughput preparation of antibodies and while removing greater than 95% of salts.recombinant proteins to facilitate the process ofFor further support call PhyNexus at 408.267.7214 or e-mail further information on PhyNexus products visit our website at Copyright © 2008, PhyNexus, Inc., All Rights Reserved
  2. 2. PhyNexus PhyTip 5K Gel Filtration Technical Note Fig 1 PhyTip 5K Gel Filtration Columns The design of the patent pending 5K gel filtration columns (Fig 1) is based upon a unique manufacturing process where pipette tips are filled with gel filtration resin. Thin screens are placed above and below the resin bed that retain the gel filtration media within the column structure, while allowing the passage of liquids. The top frit is a larger liquid interface barrier that helps facilitate the transfer of liquid to the top of the column. PhyTip 5K gel filtration columns retain small molecules while molecules over 5 KDa will pass through the gel filtration media. screens Desalting or Buffer Exchange Process Using the MEA Personal Purification System, a row of either 200µL orResin bedresin bed PhyTip 5K gel filtration columns are conditioned in 1mL of buffer. After the columns areconditioned, samples are added and processed followed by differing volumes of chaser buffer additionand processing. The samples can be collected in fractions to determine the optimal sample recoveryand salt removal conditions. Using the MEA Personal Purification System, each row of 12 samplesrequires 30 minutes to process while the entire plate of 96 samples can be processed in 2.5 hours.ResultsThe process of desalting or buffer exchange using the PhyTip 5K desalting columns and the MEAPersonal Purification System have been carefully optimized to ensure both maximum recovery of theprotein of interest and removal of salt. 160 Fig 2A 140Consistent Volume Recovery 120 Exp 1 vol in uLMost pharmaceutical companies streamline the 100 80 Exp 2processes of antibody/protein production, 60 Exp 3purification, and desalting/buffer exchange for 40cell-based assays. In order to efficiently process 20 0the samples from production to cell-based assays, 1 2 3 4 5 6 7 8 9 10 11 12high throughput solutions are desirable. PhyTip columnsHowever, in practice, achieving a true highthroughput solution requires a level of precision Fig 2B Chaser Vol E1: (80uL) E2: (80uL) E3: (130uL)not easily achieved. High throughput sample Column1 73.4 70.9 122.6preparation is not advantageous if subsequent Column2 71.4 77.4 132.6quantification is required prior to the next assay. Column3 65.9 64.4 112.6The solution requires consistent mass and Column4 61.8 65.2 115.0concentration recovery. Column5 61.6 73.8 118.8 Column6 61.9 61.6 106.5To determine the consistency of volume Column7 60.0 73.6 119.4 Column8 71.4 76.7 120.6recovery from 200µL resin bed PhyTip gel Column9 67.1 72.6 133.4filtration columns, 80µL or 130µL of buffer was Column10 61.6 71.6 121.0loaded on top of 11 or 12 conditioned columns. Column11 64.7 70.7 110.0Flow through was collected and the volume was Column12 65.4 74.9measured. In three replicate experiments, Average 65.5 71.1 119.3recovered volumes varied by less than 10% CV SD 4.5 5.0 8.4(Fig 2A & 2B). CV 6.9 7.0 7.0For further support call PhyNexus at 408.267.7214 or e-mail further information on PhyNexus products visit our website at Copyright © 2008, PhyNexus, Inc., All Rights Reserved
  3. 3. PhyNexus PhyTip 5K Gel Filtration Technical NoteSeparation by PhyTip gel filtration column Fig 3Sample (far left microfuge tube) containing brownmyoglobin protein (16.7kDa) and yellow DNP- 1 2 3 4 5 6 7 1glutamate salt (313Da) was loaded onto a 600uLPhyTip gel filtration column. The same PhyTip columnat different steps of desalting is pictured along withthe collected flow through. From left: 1) columnconditioned by PBS buffer prior to sample loading, 2)column after 200µL sample has entered the resin bed,3-6) column after 100µL PBS buffer is applied, 7)column after a final chase of 400uL PBS buffer. Thisexample illustrates a typical separation allowing the 2 3 4 5 6 7user to discard sample flow through (2) to maintainconcentrated purifications. Additionally, salt isretained until fraction (6), while most of the protein isreleased by fraction (4).HPLC chromatogram (at right) of His-Tagged Sample Pre-Desalting Fig 4ubiquitin at 0.05mg/mL concentration spiked with250mM imidazole. 300µL samples (red) were appliedto 600µL columns. The resulting desalted sample was His-Ubiquitincollected and adjusted to 300µL with water (blue) tocompare with the starting sample. Water was injectedas a blank (green). The Post-desalting sample shows Sampleboth recovery of protein and complete removal of Post-Desaltingimidazole. BlankMyoglobin recovery and salt removalAfter columns are conditioned, samples are first added and processed followed by different volumes ofchaser buffer addition and processing. The samples can be collected in fractions to determine themaximum sample recovery. In this experiment, we used myoglobin and DNP-glutamate (20µL samplevolume) and separated myoglobin from DNP-glutamate using 80µL chaser volume (Table 1) and 200µLresin bed gel filtration columns. As shown in the table below, we were able to recover ~80% ofmyoglobin and remove ~99.8% of DNP-glutamate. Using the MEA Personal Purification System eachrow of 12 samples can be separated in about 30 minutes. A full plate of 96 samples takes about 2.5hours to process. Table 1: A364 A409 Volume pmol Myo mol Salt % prot rec %salt rem Myoglobin input 1.165 20.0 47843.9 Myoglobin sample1 0.205 90.5 38095.5 79.6 Myoglobin sample2 0.200 94.8 38932.2 81.4 DNP-glutamate input 2.440 20.0 70469.3 DNP-glutamate sample1 0.003 88.7 96.1 99.9 DNP-glutamate sample2 0.006 89.3 193.4 99.7For further support call PhyNexus at 408.267.7214 or e-mail further information on PhyNexus products visit our website at Copyright © 2008, PhyNexus, Inc., All Rights Reserved
  4. 4. PhyNexus PhyTip 5K Gel Filtration Technical Note uL/% recov mg/mL Fig 5IgG separation and recovery 140IgG was separated from free dye using a 200µL 120 1.2resin bed gel filtration column. In this experiment, 100 1.0 Vol Rec ( uL)PhyTip gel filtration columns were first conditioned 80 Conc [ mg/ mL]with 800µL of PBS. After conditioning, 40µL of 60 %IgG r ec[2mg/mL] sample was added and processed 40 0.4followed by 130µL of chaser (PBS) buffer to the 20 0.2PhyTip gel filtration column to collect and separate 0 1 2 3 4 5 6IgG from unlabeled dyes.Figure 5 and Table 2 demonstrate that we were able to recover an average of 76% of IgG (Table 2, Fig.5 red) with an average of 0.5mg/mL concentration (Table 2, Fig. 5 blue) in an average final volume of121µL (table 2, fig 5 black). As shown in Table 2, the percent IgG recovery, concentration, and finalrecovered volume had very small CV values. These SD and CV values show the reliability andconsistency of the PhyTip gel filtration columns. Table 2 Column Final Volume Concentration Mass Recovered % Protein # [µL] [mg/mL] (mg) Recovered 1 122 0.49 0.060 75 2 119 0.50 0.060 74 3 122 0.50 0.061 76 4 119 0.54 0.064 80 5 122 0.48 0.059 73 6 123 0.51 0.063 78 Ave 121 0.50 0.061 76 SD 2 0.02 0.002 3 CV 1.4 4.1 3.5 4ConclusionPhyTip 5K gel filtration columns and the MEA Personal Purification System represent a high throughputsolution for rapid desalting and buffer exchange. PhyTip 5K gel filtration columns can process 12samples in 30 minutes compared to the laborious and time consuming dialysis procedure. Using PhyTip5K gel filtration columns, researchers can save hours while obtaining a high level of performance.Unlike spin columns, PhyTip columns simulate traditional chromatography and can performfractionation of protein samples. This allows researchers to optimize maximum protein recovery andsalt removal in an automated, high throughput manner.PhyTip 5K gel filtration columns can be used in combination with PhyTip purification columns on theMEA Personal Purification System. This allows researchers to combine purification with desalting andbuffer exchange. For example, 6-His-tagged proteins can be purified by PhyNexus IMAC PhyTipcolumns and eluted using high concentrations of imidazole, which can be removed by PhyTip gelfiltration columns. These processes allow researchers to perform multiple sample processing steps in ahigh throughput, walk-away scenario.For further support call PhyNexus at 408.267.7214 or e-mail further information on PhyNexus products visit our website at Copyright © 2008, PhyNexus, Inc., All Rights Reserved